FMD virus is a picornavirus which affects cloven-footed animals. It is one of the most infectious and easily transmitted diseases of livestock. The disease is generally characterized by vesicular lesions on the feet and mouth of the infected animal. Deterioration in body condition generally lowers production of animal products by 25%. Epizootics of FMDV cause major economic losses in the production and marketing of meat, dairy products, wool, hides and other commodities. For example, during an epizootic in Great Britain in 1967-68 nearly 500,000 infected or exposed cattle, swine, sheep and goats were destroyed in the process of eradicating the disease.
There are seven distinct serotypes of FMD virus. These are the European or classical types O, A and C, the Southern African Territories types SAT1, SAT2 and SAT3 and the Asian type I. E.g., K. J. H. Robson et al., "An Assessment By Competition Hybridization Of The Sequence Homology Between The RNAs Of The Seven Serotypes Of FMDV", 37 J. Gen. Virol. 271-76 (1977). Types O, A and C have been found in Europe, South America, Asia and the northern part of Africa, although the range is extending. The three Southern African Territories types were first detected in Southern Africa, but again the disease type is spreading in geographic location. The Asian type occurs in Asian countries from the Eastern Mediterranean to the Far East. Each of these serotypes is also comprised of several serological subtypes.
Vaccines to protect animals from FMDV are available. Most commonly these vaccines comprise inactivated or attenuated whole virus. They are administered under known schedules and regimes on an annual or quarter-annual basis. Because the seven virus types display a lack of cross-immunity (Robson et al., supra) and different areas of the world have a different spectrum of virus types, vaccination is sometimes carried out with a bivalent or trivalent material. However, this is not always necessary or advisable.
Production of FMD whole virus vaccines is beset by several major difficulties. First, because of the infectious character of the virus, laboratory growth of virus for vaccines must be done in isolated facilities under high containment. Second, the virus-based vaccines often display an unacceptable variation in potency after production and inactivation. Third, the vaccines must be tested under very controlled conditions to insure proper efficiency of attenuation or inactivation. Otherwise, the vaccine may cause accidental active infection or subacute progressive disease in the treated animals. All of these production problems result in higher costs for the ultimate vaccine. In addition to the above-described production problems, the use of whole-virus vaccines results in a small, but a significant, number of allergic side reactions in the treated animals. These undesirable side effects are probably caused by the many irrelevant antigenic determinants of the viral and non-viral proteins that usually contaminate viral vaccines.
One approach to avoid some of the problems inherent in the production and use of whole virus vaccines is to employ viral subunit vaccines comprising the capsid proteins of FMD virus. E.g., U.S. Pat. No. 4,140,763; H. L. Bachrach et al., "An Experimental Protein Vaccine For Foot-And-Mouth Disease", in 10 Perspectives in Virology 147-59 (M. Pollard ed. 1978).
FMD virus is characterized by four capsid proteins, identified as VP1, VP2, VP3 and VP4. The capsid proteins of FMD virus collectively protect the viral ribonucleic acid ("RNA") core of the virus against various inactivating agents. The neutralizing antigen of FMDV seems to be embodied in the VP1 polypeptide (VP3 in United States terminology) (H. L. Bachrach et al., "An Experimental Subunit Vaccine For Foot-And-Mouth Disease", International Symposium On Foot-And-Mouth Disease, Lyon 1976, 35 Develop. Biol. Standard 155-60 (1977)). Moreover, it has been reported that the antigenic portion of VP1 appears to reside in the last 1/3 of the protein, i.e., nearest its COOH terminus (R. Franz et al., "Localization And Characterization Of Two Immunogenic Regions On The Coat Protein VP.sub.Thr Of Foot-And-Mouth Disease Virus (FMDV) Subtype O.sub.1 K Inducing Neutralizing Antibodies", Munich (December 1980)). It has also been reported that two enzyme-sensitive areas of VP1 appear to exist--between sequence positions 138-154 and between positions 200-213 [K. Strohmaier et al., "Localization And Characterization Of The Antigenic Portion Of The FMDV Immunizing Protein", presented at the "Positive Strand Virus" session at the meeting of the Society for General Microbiology, Cambridge (April 1981)]. VP1 has been purified and employed to vaccinate swine against challenges by virus (H. L. Bachrach et al., supra). However, at least 10 times more protein than virus-based vaccines were required to effect immunization. Therefore, two or more vaccinations with the VP1 protein-based subunit virus are usually required to protect an animal from FMD virus.
The use of subunit vaccines eliminates to some extent antibody formation against the many irrelevant antigenic determinants of the viral and non-viral proteins that often contaminate viral vaccines. Their use may therefore lessen the possible side effects of viral vaccines. Further, the subunit vaccines are devoid of viral nucleic acid and therefore presumably without risk of causing active infection or subacute progressive disease in the treated herds. However, while these subunit vaccines avoid some of the problems which characterize whole virus-based vaccines, there are also disadvantages in their use. First, to obtain the capsid protein, virus must be cultured and grown. Therefore, the isolated facilities and high production containment attendant to FMD virus growth are not avoided. Second, the proteins must be separated from the virus and highly purified. This process is both slow and expensive. Moreover, if the proteins are not sufficiently purified, the resultant vaccine may still contain enough virus to cause accidental infection or subacute disease.
To avoid the problems that disadvantage the above-described VP1-based vaccines, recombinant DNA technology has also been employed to produce recombinant DNA molecules characterized by a DNA sequence or fragment thereof coding for a polypeptide displaying FMDV antigenicity. This work, conducted jointly by Biogen N.V. and the Max-Planck-Institute for Biochemistry, is described in European patent application No. 40,922. Using the recombinant DNA molecules described in that application, FMD viral-specific nucleotide sequences and FMDV antigenic polypeptides were produced without the necessity of growing large amounts of virus, purifying proteins from the virus or inactivating or attenuating the virus. The antigenic polypeptides and DNA sequences described in that application are useful in compositions and methods for the treatment of FMD virus infection.
Vaccines against FMDV that are based on bacterially-made VP1, accordingly, are much advantaged over vaccines that are based on live virus or proteins isolated from live virus. However, such bacterially-made VP1-based vaccines may be even further improved by replacing the complete VP1 active component in those vaccines with an active component comprising substantially only the antigenic portion of VP1. Such modification of the vaccine results in several compositional and process advantages. The vaccine will contain only a single, or at most very few, antigenic sites. The substantial absence of non-FMDV specific determinants will further reduce the possibility of allergic side reaction in treated animals. The use of smaller peptides will also permit manufacture of a vaccine that will contain a much higher ratio of active component to weight than the previous whole VP1-based vaccines. And, if the antigenic portion of VP1 is prepared by chemical synthesis, its purification will be easier and the resulting vaccine will be a non-biological product. Finally, the smaller peptides may be more easily modified in composition and conformation than the former polypeptides so as to improve further the activity of those polypeptides and the vaccine based upon them.
The potential of identifying and preparing immunologically-active small peptides has been demonstrated. E.g., F. A. Anderer et al., "Properties Of Different Artificial Antigens Immunologically Related To Tobacco Mosaic Virus", 97 Biochim. Biophys. Acta 503-09 (1964); H. Langbeheim et al., "Antiviral Effect On MS-2 Coliphage Obtained With A Synthetic Antigen", 73 Proc. Natl. Acad. Sci. USA 4636-40 (1976); F. Audibert et al., "Active Antitoxic Immunization By A Diphtheria Toxin Synthetic Oligopeptide", 289 Nature 593-94 (1981); E. H. Beachey et al., "Type-specific Protective Immunity Evoked By Synthetic Peptide Of Streptococcus pyogenes M Protein", 292 Nature 457-59 (1981); and G. M. Muller et al., "Anti-Influenza Response Achieved By Immunization With A Synthetic Conjugate", 79 Proc. Natl. Acad. Sci. USA 569-73 (1982).
In addition, the amino acid sequences of antigenic polypeptides determined using recombinant DNA technology have been employed to predict areas of possible antigenicity. The peptides defined by those areas have then been synthesized and their immunological characteristics analyzed. For example, the amino acid sequence of the surface antigen of hepatitis B virus predicted from the nucleotide sequence of the gene coding for that antigen was analyzed to predict the internal and external residues of the antigen and a series of peptides were prepared based on those predictions. R. A. Lerner et al., "Chemically Synthesized Peptides Predicted From The Nucleotide Sequence Of The Hepatitis B Virus Genome Elicit Antibodies Reactive With The Native Envelope Protein Of Dane Particles", 78 Proc. Natl. Acad. Sci. USA 3403-07 (1981); T. P. Hopp, "A Synthetic Peptide With Hepatitis B Surface Antigen Reactivity", 18 Molec. Immun. 869-72 (1981); G. R. Dreesman et al., "Antibody To Hepatitis B Surface Antigen After A Single Inoculation Of Uncoupled Synthetic HBsAg Peptides", 295 Nature 158-60 (1982); European patent application No. 44,710.
A similar analysis and synthesis has also been described in European patent application No. 44,710 for preparing small peptides as antigens against FMDV subtype O.sub.1 K. As a result of that analysis, five peptides were reportedly prepared--peptide 1: amino acids 1-18 of VP1; peptide 2: amino acids 9-24; peptide 3: amino acids 17-32; peptide 4: amino acids 25-41; and peptide 5: amino acids 21-41. See, e.g., C. Kurz et al., "Nucleotide Sequence And Corresponding Amino Acid Sequence Of The Gene For The Major Antigen Of Foot And Mouth Disease Virus", 9 Nucleic Acids Research 1919-31 (1981). These peptides were stated to carry the specific antigenic determinants of FMDV (European patent application No. 44,710).
In addition, the amino acid sequence of VP1 determined by recombinant DNA technology has been employed with an enzyme cleavage pattern for natural VP1 of FMDV subtype O.sub.1 K. As a result, a series of VP1-derived fragments were localized along the protein (Strohmaier et al., supra). The reported fragments were stated to comprise the following amino acid sequences of natural VP1 of FMDV subtype O.sub.1 K: 1-9, 10-36, 1-138, 1-145, 37-54, 55-180, 146-213, 155-200 and 181-213. Only fragments 55-180, 146-213, and 181-213 were said to induce neutralizing antibodies. Accordingly, the sequence positions between 138-154 and between 200-213 were predicted to be the antigenic portions of VP1 of FMDV subtype O.sub.1 K, because those were the only regions in antibody inducing agents not overlapped by non-inducing areas (Strohmaier et al., supra).